d-Limonene is a natural component of a variety of foods and beverages and is found in many fruits, vegetables, meats, species and other food items. Recently NTP found conducted chronic two-year studies of d-Limonene in rats and mice and found there was clear evidence of carcinogenic activity for male F344 rats only as shown by increased incidences of tubular cell hyperplasia, adenomas and adenocarcinomas of the kidney. The response observed in male rats may be linked to specific renal perturbation of alpha 2U-globulin, unique to the male rat kidney. This study was performed to evaluate the hyaline droplet formation and the presence of alpha 2U-globulin was determined by the enzyme linked immunosorbent assay (ELISA) in the kidney homogenates. Total protein were measured in the aliquots for alpha 2U-globulin by the Lowry method. We have confirmed that d-Limonene produced significant dose-related increase of alpha 2U-globulin only in male rats. These results suggest that d-Limonene associated nephrotoxicity in male rats may be related to altered catabolism of alpha 2U-globulin, a low molecular weight protein synthesized by the liver under androgenic control. Thus we developed the ELISA technique for alpha 2U-globulin and refined the procedures for our use in the future research projects at NTP and NIEHS. (These results were published in the Toxicologist, 1990.)